Method for exposing peptides and polypeptides on the cell surface of bacteria

ABSTRACT

The inventive method allows peptides or polypeptides to be exposed on the surface of gram-negative host bacteria using specific intimin-based anchor modules. Intimins with shortened carboxy terminals have been found to be particularly suitable anchor modules for passenger domains in the exterior  E. coli  cell membrane. According to the method, host bacteria are transformed using vectors, on which are located a fused nucleic acid sequence consisting of a sequence segment which codes for an intimin with a shortened carboxy terminal and a nucleic acid sequence segment which codes for the passenger peptide that is to be exposed. The invention permits a particularly large number of passenger domains to be exposed on the cell surface of the bacteria, without adversely affecting the viability of the bacteria.

The present invention concerns a method for exposing peptides, includingpolypeptides and proteins, depending on the length of the sequenceexposed, on the surface of Gram-negative bacterial cells, especiallyEscherichia coli cells, an accompanying process for producing a variantpopulation of surface-exposed peptides or polypeptides and foridentifying bacteria which carry peptides or polypeptides with aparticular desired property, and vectors and host bacteria which can beused in the process.

In general, the method allows expression of recombinant proteins, whichcan be receptors or ligands, on the bacterial surface and selectionbased on affinity of binding to a binding partner. The method allowsexpression of peptide and polypeptide libraries on the surface ofbacterial cells, by means of which peptide molecules with high affinityto a ligand can be identified. The method allows exposure of aparticularly large number of passenger proteins or peptides on thesurface of a bacterial cell without adversely affecting its cellularviability. This method further allows setting the number of moleculespresented on the surface of a cell as desired.

In particular, the present invention also concerns isolation ofmonospecific antibodies from polyclonal antibody mixtures by binding topeptide epitopes anchored to the cell surface.

BACKGROUND

Expression of proteins and protein domains on the surface ofself-replicating carriers such as bacteriophages, bacteria, yeasts,etc., is currently under intense investigation. The primary objective isto achieve coupling of the functional expression of a property of theprotein exposed on the surface of the carrier (phenotype) with thefundamental genetic coding (genotype). Examples of successfulphenotype/genotype coupling appear, among others, in the use of yeastsas carriers of surface-exposed proteins. For instance, a molecularlibrary of variant yeast cells is generated, each one of which hasdifferent immunoglobulin fragments exposed on its cell surface. Thosecells which exhibit increased affinity to a specific ligand molecule canbe isolated from that library of antibody variations (Boder and Wittrup(1997), Nat. Biotechnol. 15:1553).

Bacteria have broad applications for cell surface exposure. BothGram-positive and Gram-negative types are used. For instance, proteinscan be exposed on Staphylococcus xylosus and Staphylococcus carnosus byStaphylococcus aureus protein A. Enzymes can be anchored to the surfaceof S. carnosus by fusion to Staphyloccus aureus fibronectin bindingprotein B (FnBPB) (Strauss and Götz (1996), Mol. Microbiol. 21:491–500).However, Gram-positive bacteria are less suitable for exposure of largepeptide libraries because it is difficult to introduce the correspondinggene variants into the cells by transformation and to generate asufficiently large number of independent clones which differ withrespect to the coding nucleotide sequence of the surface-exposed proteinvariants.

Gram-negative bacteria, on the other hand, are very well suited togeneration of molecular libraries and to the accompanying exposure ofaltered proteins because of their high transformation yield (>10° permicrogram of plasmid DNA for E. coli), and so are preferred hostorganisms.

Various systems have been described for exposing recombinant proteins onthe cell surface of Gram-negative bacteria (Georgiou et al. (1997),Nature Biotechnol. 15:29–34). In general, surface exposure is attainedby fusing gene segments of a bacterial surface protein with the gene forthe protein to be exposed. The proteins usually used as carriers arethose which are secreted and/or localized in the external membrane ofGram-negative bacteria and therefore contain the signals needed fortranslocation through the cytoplasmic membrane, passage into thebacterial periplasm, and integration into the external membrane oranchoring on the surface of the external membrane. The carrier proteinsthat have been used most are those which are themselves integralcomponents of the external membrane of E. coli. Those include, amongothers, PhoE (Agterbert et al. (1987), Gene 59:145–150) or OmpA(Francisco et al. (1992), Proc. Natl. Acad. Sci. USA 89:2713–2717); butthere are disadvantages to their use. For instance, protein sequencescan be inserted only into surface-exposed loops of these proteins. Thatresults in conformationally fixed amino and carboxy terminations, anddrastically limits the length of the peptide sequence to be inserted.Use of the peptidoglycan-associated lipoprotein (PAL) as a carrierprotein does indeed result in transport to the external membrane, but itis impossible to expose active and correctly folded protein sequences onthe surface of E. coli (Fuchs et al. (1991), Bio. Technology 9,1369–1372). It has been possible to expose large proteins on the surfaceby (a) use of fusion of a fragment of the Escherichia coli Lpp and ofthe OmpA protein as the carrier protein portion, to the carboxy end ofwhich the passenger protein sequence is attached (Francisco et al.(1992), Proc. Natl. Acad. Sci. 89:2713–2717); (b) use of the IgAprotease (domain (IgAβ) and other bacterial autotransporters (Maurer etal. (1997) J. Bacteriol. 179: 794–804), and (c) by use of the icenucleation protein of Pseudomonas syringae (InaZ) (Jung et al. (1998),Nature Biotechnol. 16:576–580 as the carrier protein portion.

It is clear from the examples above that proteins can be exposed on thebacterial cell surface by joining a passenger domain to a carrierprotein by fusion of the corresponding coding DNA sequence with thecoding sequence of a selected protein of the external membrane ormembrane protein fragment. In this case the membrane protein or membraneprotein fragment provides the force needed for the membrane localizationand anchoring. Here the carrier protein of the external membrane should(a) have a secretion signal that assures passage through the cytoplasmicmembrane; (b) exhibit a localization signal for embedment into theexternal membrane; (c) appear on the cell surface in the highestpossible number of copies; and (d) not have a negative effect on thestructural and functional integrity and, in particular, the vitality ofthe host cell.

Substantial problems have been found, though, with the processesdescribed at the state of the art for production of heterologouspassenger proteins using proteins of the external membrane, particularlywith respect to requirements (c) and (d). A high expression ratio and ahigh net accumulation in the external membrane are always accompanied byhigh mortality of the bacterial cells which expose them. For instance,strong over-expression of fusion proteins with Lpp-OmpA as the membraneanchor is lethal (Daugherty et al. (1999), Protein Eng. 12:613–21). Highcell mortality was likewise described for use of the autotranporter ofthe IgA protease (IgAβ) (Wentzel et al. (1999), J. Biol. Chem. 274:21037–21043). Jung et al. Introduced the ice nucleation protein ofPseudomonas syringae, which is a glycosyl-phosphatidylinositol anchoredprotein of the external membrane, into E. coli as a carrier protein forcell surface exposure of passenger proteins (Jung et al. (1998), NatureBiotechnol. 16: 576–580). This carrier protein does allow stableexposure of passengers on the surface of the external membrane; but thefusion proteins aggregate in clusters on the bacterial surface. Thatcharacteristic is undesirable for the purpose of selecting peptides andpolypeptides with high affinity to a specific binding partner.

Aside from the proteins integral to the external membrane, other surfacestructures present on the cell surface, such as flagellae, pili,fimbriae, etc., have been used as carriers for exposure of passengerdomains. Various peptides of Hepatitis B virus were stably expressed andexposed on the bacterial surface by use of flagellin, a subunit of theflagellum, as the carrier (Newton et al. (1989), Science 244: 70–72).However, as for use of fimbrin as a structural carrier protein, exposureof passenger domains remains limited to small peptides (Hedegaard et al.(1989), Gene 85: 115–124).

Technical Problems, and Their Solution by the Present Invention

The present invention is, therefore, based on the technical problem ofproviding carrier proteins, which do not result in the disadvantagesstated above, especially with use of Escherichia coli.

An optimal presentation procedure must meet the following requirements:

-   1. The peptide/protein to be exposed should preferably be anchored    on the surface of a bacterial cell in the highest possible number of    copies.-   2. The peptide/protein exposure should not impair viability.-   3. The number of peptide/protein molecules exposed on the surface    per cell should be controllable within wide limits.

No method of bacterial surface exposure which meets these requirementsin all points has yet been described.

SUMMARY OF THE INVENTION

To achieve the objective stated above, a process is provided forexposure of peptides and/or polypeptides on the surface of host bacteriain which one (a) produces a Gram-negative host bacterium which istransformed with a vector to which is localized a fused nucleic acidsequence that (i) has a sequence segment that codes for an Intiminshortened by at least the C3 domain at the carboxy terminus region asthe anchoring domain and

-   (ii) has a nucleic acid segment coding for the passenger peptide    and/or passenger polypeptide to be exposed, and (b)-   cultivates the host bacterium under conditions in which the coded    nucleic acid-   sequence is expressed and the peptide or polypeptide coded by the    nucleic acid-   sequence (ii) is exposed on the surface of the host bacterium, such    that the nucleic acid sequence (ii) is heterologous with respect to    the nucleic acid sequence segment coding for the Intimin membrane    anchoring domain.

The shortened Intimin can be shorted by at least one more of the domainsD0, D1 and/or D2 in the carboxyterminal region of 280 amino acids (asidefrom the shortening by the D3 domain).

The method of the invention allows exposure of peptides or polypeptideson the surface of Gram-negative host bacteria using certainIntimin-based anchoring modules. It has been found that Intiminsshortened at the carboxy terminus are particularly well suited asanchoring units in the external E. coli cell membrane for passengerdomains.

The invention is based on providing a gene construct in which a specialcarrier protein (a fragment of the “Intimin”, see below) is used as theexposure anchor. To expose a specified peptide/protein, the coding geneis fused to the coding sequence of the Intimin gene fragment in thecontinuous reading frame. Surprisingly, it is found that when an Intiminfragment is used as the membrane anchor a very large number of moleculescan be exposed in the bacterial membrane without impairing the viabilityof the host bacteria.

(b) [sic/tr. note #1] We have succeeded in establishing the number ofmolecules per cell as we wish by combined regulation of the expressionof the Intimin gene at the transcription and translation level.Processes previously described for regulating gene expression for thepurpose of establishing surface-exposed molecules do not do that.

Intimin as the Exposure Anchor

The further development of the invention provides that the Intiminanchoring domain in the external bacterial membrane be derived from thegenus of the Enterobacteriaceae and used in a host bacterium of a genusof the Enterobacteriaceae [sic/tr. Note #2]. It is further preferablefor the anchoring domain to be a fragment of the Intimin ofenterohemorrhagic E.coli or a variant of it. At present acarboxy-terminal shortened variant of the BacA Intimin fromenterohemorrhagic Escherichia coli O157:H7 comprising amino acids 1 to659 (see FIG. 16A, SEQ ID NO: 25) or, alternatively, amino acids 1 to753 (see FIG. 16B. SEQ ID NO: 26), is particularly preferred.

Many strains of pathogenic bacteria have surface structures such aspili, glycoproteins and other proteins, such as homopolymeric andheteropolymeric carbohydrate glycocalices, by means of which thebacteria adhere to surfaces of eucaryotic cells. Proteins of thebacterial cell surface called Intimins play an important part in thefirm adhesion of enteropathogenic (EPEC) or enterohemorrhagic E. coli(EHEC) bacteria to the surfaces of eucaryotic host cells. TheIntimin-mediated adhesion allows the bacteria to multiply on thesurfaces of the colonized host cells. Intimin is a member of the familyof bacterial adhesion molecules which have sequence homologies with eachother in the area of the amino-terminal region (McGraw et al. (1999),Mol. Biol. Evol. 16:12–22).

Intimin is the product of the Eae gene. It has 939 amino acid groups.Its cell-binding activity is localized in the 280 C-terminal amino acidgroups. Intimin is anchored in the external membrane with itsamino-terminal domain, which exposes the 280 carboxy-terminal aminoacids. These 280 groups code for three domains, two immunoglobulin-likedomains and one lectin-like domain (Kelly et al. (1999), Nat. Struct.Biol. 6:313–318) code for these 280 groups, which are responsible forbinding to eucaryotic cells. The structures of domains D1, D2 and D3 areknown, but not the structure of the amino-terminal domain (Kelly et al.(1999) Nat. Struct. Biol. 6:313–318). FIG. 1 shows the schematicstructure of Intimin and its anchoring in the external membrane.Corresponding domains of other Intimins can be derived from sequencecomparisons.

Except for the 200 amino-terminal amino acid groups, Intimins exhibitsequence homologies with invasins from Yersinia and other bacteria whichmake it possible for the bacterium to enter cultivated mammalian cellsthrough binding with integrins (Leong et al. (1990), EMBO J.9:1979–1989). Integrins and invasins do, to be sure, have similarsequences, but they are assigned to different protein families withrespect to their functions (Batchelor et al. (2000), EMBO J.19:2452–2464).

Surprisingly, surface presentation of peptides or polypeptidesdistinctly improved over the state of the art was attained by use of afragment of Escherichia coli Intimin as the transporter domain forbacterial surface localization of passenger proteins and passengerpeptides, particularly also of short synthetic peptides having lengthsof preferably 6 to 20 amino acids, by disulfide-bridged peptides andpolypeptides, especially oligopeptides on the structural basis of thecystine node folding motif (Pallaghy et al. (1994), Protein Sci. 3:1833–1839), or by bacterial (e. g.: (-lactamase inhibitor protein) andeucaryotic polypeptides (e. g., interleukin 4).

Identification of Eae Intimin as the Carrier Protein for SurfaceExposure of Passenger Proteins

The Escherichia coli Intimin that is used preferably is localized in theexternal membrane of Escherichia coli. It naturally exposes at least oneprotein domain on the outer side of the external membrane. TheEscherichia coli Intimin is anchored in the external membrane, andcarries at its carboxy-terminal end four domains which are necessary forinteraction with a receptor protein on the surface of epithelial cells.

Beginning with the working hypothesis that substitution of at least oneof the Intimin domains exposed on the cell surface by a heterologouspassenger domain would result in that domain being exposed on thesurface, a gene fusion was produced from a nucleic acid segment codingfor a passenger protein and one coding for an Intimin fragment. Example1 presents the gene fusion from the Intimin fragment and the passengerdomain, the vector construction, and the expression in host bacteria,compared with other surface presentation methods.

In general, the Intimin gene or gene fragment selected for gene fusionis amplified by the polymerase chain reaction and cloned in a vectorsuitable for expression in the intended host bacterium. A gene for apassenger peptide to be exposed is introduced into the same genedownstream from the selected Intimin fragment in the same reading frame.The vector also contains an exogenously inducible promoter operativelylinked with the Intimin gene and with the passenger peptide fused withit. Other functional sequences, such as marker genes, can also bepresent.

In further development of the invention, the process is arranged so thatthe expression of the fusion gene from the nucleic acid sequence segmentcoding for the shortened Intimin and for the passenger protein can beregulated by (i) replacing a codon coding for a glutamine in theshortened nucleic acid sequence segment coding for the shortened Intiminby an amber stop codon (TAG), and (ii) using an Escherichia coli hoststrain in which a translation of the mRNA of the fusion gene isaccomplished by providing a controllable quantity of suppressor tRNA,which allows over-reading of the stop codon on translation. Thisprocedure allows effective regulation of the expression of the segmentexposed within the host cell, and is useful for practically all knownexposure processes.

In the further developed process a CAG codon within the Intimin fragmentis replaced by a TAG stop codon. That is accomplished, for instance, bycloning a PCR fragment in which the CAG codon #35 in the Intimin isreplaced by a TAG stop codon. By use of an amber suppressor strain, thisstop codon is over-read and a glutamine group is incorporated at thisposition. As the efficiency of amber suppression is low, fewer moleculesare synthesized than in the absence of the stop codon. The result isthat the number of surface-exposed molecules remains within an extentthat is tolerable for the cell (in this connection, see Christmann etal. (1999), Protein Eng. 12: 797–806). Any expression vectorparticularly usable for expression in E. coli can be used. The vectorpASK75 (see below), among others, is a suitable starting vector.

Regulation of the Gene Expression

Expression of a gene is usually regulated by the coding sequence of agene being brought under control of a promoter such that the number oftranscriptions per unit time can be regulated by the concentration of aninducer molecule added exogenously. For instance, the lac promoter, theara promoter, or the tetA promoter can be considered for that purpose(Lutz & Bujard (1997), Nucleic Acids. Res. 25:1203). It has notpreviously been possible satisfactorily to regulate gene expression withthe goal of establishing a desired number of surface-exposed moleculesper bacterial cell by controlled induction of the transcription of thegene for surface-exposed fusion proteins. Variation of the concentrationof an added inducer has not previously resulted in accumulation offusion proteins on the surface of the bacterial cell depending on theconcentration of the inducer (Daugherty et al. (1999), Protein Eng. 12:613–621). A slight improvement was gained by adjusting the netaccumulation by means of the induction period. That means that thetranscription inducer was added to a bacterial culture, and the cellswere incubated with the inducer for different times. The longer theinduction time, the higher the number of surface-exposed cells per cell[sic/tr. note #5]. This process is not suitable for highly parallelbiotechnological applications, as it requires sample collection atdifferent growth times. Also, the cells are in different physiologicalstates, depending on the growth time and the absorbance [sic/tr. note#7] attained.

The process which we have developed eliminates this disadvantage. Thegene expression is controlled on two levels, the level of transcriptionand the level of translation.

In one process according to the invention, fusion proteins are producedby replacing a codon coding for glutamine (CAG) in the nucleic acidsequence coding for the Intimin or Intimin fragment by a amber stopcodon (TAG). This codon is preferably the first glutamine codon of theamino acid sequence of Intimin or the Intimin fragment. Likewise, adifferent codon within the first 100 amino acids of the Intimin orIntimin fragment can be replaced by TAG. Now a modified E. coli strainis offered in trans a modified glutaminyl-tRNA, which carries thegenetic marker supE. This supE tRNA is able to pair in the translationwith a TAG codon, and to cause suppression of the translation stop.

An E. coli host bacterium which contains the nucleic acid sequencesegment coding for the supE gene in operative linkage with acontrollable promoter is according to the invention. The PI lacpromoter, with which the intensity of expression and thus the rate ofsynthesis of supE transfer RNA can be controlled by the amount of theinducer IPTG added to the growth medium for the host bacteria, ispreferred. In one typical example, the nucleic acid sequence segment,which codes for a supE tRNA gene in operative linkage with acontrollable promoter is localized in a vector compatible with theexpression vector. Transcription of the gene for the Intimin passengerdomain fusion protein is switched on by addition of an inducer(anhydrotetracycline in this case). Different, and freely adjustable,quantities of suppressor tRNA for synthesis of the Intimin fusionprotein are made available in the cell by varying the amount of IPTGinducer. Finally, the amount of supE tRNA determines the average numberof passenger domains exposed on the surface of a bacterial cell. FIG. 5shows schematically the newly developed expression process.

Embodiments

The process according to the invention produces a host bacteriumtransformed with one or more compatible vectors. Such a vector containsa fused nucleic acid sequence in operative linkage with a promoter andoptionally other sequences needed for the expression. This fused nucleicacid sequence includes (a) a nucleic acid sequence segment coding for anIntimin fragment which makes possible exposure of the peptide orpolypeptide coded by segment (b) on the outside of the external membraneof the host bacterium and (b) a nucleic acid sequence segment coding forthe protein and/or peptide to be exposed.

In one preferred embodiment, then, the present invention concerns anIntimin, a fragment of Intimin, or a carrier protein homologous withIntimin, which exerts a transporter function and allows surface exposureof recombinant proteins in the host bacteria in high numbers of copies.This involves the amino-terminal fragment of the Eae Intimin fromenterohemorrhagic Escherichia coli Serotype O157:H7 (Louie et al.(1993), 61:4085–4092) which extends from amino acid 1 to 659. Along withthis specific sequence, the invention also covers use of variants, whichcan, for example, be produced by alteration or deletion in the aminoacid sequence in the sequence segments not essential for translocationthrough the cytoplasmic membrane and localization in the externalmembrane.

Another typical example involves gamma Intimin from E. coli (Gene BankAccession Number AF081182), Intimin from E. coli O111 :H— (Gene BankAccession Number AAC69247) or Intimin from other Escherichia coliserotypes or the Intimin from Citrobacter freundii (Gene Bank AccessionNumber AAA23097) as the transporter protein used. The DNA sequences andthe amino acid sequences derived from them for the Intimins listed abovecan be found as NCBI citations (National Center for BiotechnologyInformation, USA) at the locations listed below.

Other Intimin domains can be derived from protein sequences indatabases, from protein sequences based on DNA sequences available indatabases, or from protein sequences determined by sequence analysisdirectly or indirectly from the DNA sequence. The corresponding codingregions (genes) can be used to produce vectors or fusion protein genes,which make possible effective surface expression of passenger proteinsin Gram-negative bacteria, especially Escherichia coli.

In the invention, surface presentation or exposure means that the fusionproteins or passenger domains are localized on the side of the externalbacterial membrane toward the medium. Surface-exposed passenger proteinsin intact Gram-negative bacteria are freely accessible for bindingpartners.

In one preferred embodiment, the present invention thus enables surfaceexposure of peptides or, in a further embodiment, the surface exposureof peptide libraries in Gram-negative bacteria, especially in E. coli,and their use to determine affinity to an antibody or another receptor.

In another preferred embodiment, the present invention makes possiblemapping of epitopes and isolation of monospecific antibodies from anantibody mixture. Epitope mapping means that the peptide with thehighest affinity to an antibody or another receptor, exposed on thesurface of the producing strain, is identified. That makes clear acritical advantage of the present invention for expression of peptidelibraries, compared with the phage systems used for such applications(Makowski (1993), Gene 128: 5–11; Kuwabara et al. (1997), Nat.Biotechnol. 15: 74–78). In the bacterial system according to theinvention, selection of the clonal producers occurs simultaneously withidentification of a peptide having the desired binding property. Theycan be multiplied immediately. In one typical example, clonaldescendants of the producers were used to purify or isolate an antibodyor another receptor from a mixture of molecules. That occurs throughbinding of the receptor molecules with high affinity to the peptidemolecules exposed on the bacterial surface, followed by separation ofthe unbound molecules by centrifugation and/or filtration, andseparation of the monospecific antibodies or receptor molecules from thesurface-exposed peptides.

Multiplication of the strain expressing the desired surface-exposedpeptide or protein accomplishes amplification of the correspondingcoding gene. Sequence analysis of that gene allows unambiguous andsimple identification and characterization of the peptide or protein.Thus a peptide library prepared in that manner contains fusion proteins,made up of an Intimin or an Intimin fragment and a peptide or proteinproduced and surface-exposed in a Gram-negative bacterium, preferably E.coli. In one typical example, cloning of synthetic oligonucleotidesdegenerated at selected positions behind the coding sequence of Intiminor Intimin fragments achieves the high variance of the differentexpressed proteins.

In one particularly preferred embodiment, the process according to theinvention makes possible surface expression and variation of a peptideor polypeptide having an affinity to a binding partner, a ligand, areceptor, an antigen, a protein with enzymatic activity, an antibody, oran antigen-binding domain of an antibody.

The process according to the invention for producing a variantpopulation of surface-exposed peptides and for identification ofbacteria, which carry the peptides or polypeptides with a desiredproperty, is organized in the following steps:

-   1) Production of at least one fusion gene by cloning the coding    sequence of a desired passenger in the continuous reading frame    downstream from an Intimin gene or an Intimin gene fragment in at    least one vector.-   2) Variation of the passenger by cloning passengers from a gene    mixture, or through site-directed mutagenesis, e.g., by the    polymerase chain reaction (PCR) using oligonucleotides with    deliberately replaced bases, by random mutagenesis using    oligonucleotide mixtures with randomly produced base sequences in    selected sequence segments in the PCR, by error-prone PCR, by    randomly controlled chemical mutagenesis, or by use of high-energy    radiation.-   3) Incorporation of the vector or vectors in host bacteria.-   4) Expression of the fusion gene in the host bacteria, which then    express the fusion protein stably on their surfaces.-   5) Cultivation of the bacteria in liquid culture or on agar plates    for clonal expansion.-   6) Optional selection of the bacteria which carry the passenger with    the desired properties, and-   7) Optional characterization of the selected passenger through    sequencing of the nucleic acid sequence segment coding the passenger    peptide or polypeptide.-   8) Optional isolation and purification of a binding partner for the    passenger with the optimal properties.

This process can be carried out repetitively.

In one preferred embodiment of this process, the bacteria having astable exposed fusion protein with the desired properties are isolatedby binding to an immobilized and/or labeled binding partner, e. g., amatrix-fixed binding partner, a magnetic-particle-labeled bindingpartner, or a chromogenically or fluorogenically labeled bindingpartner.

Bacterial Surface Exposure of Protein Fragments, Epitope Mapping andIsolation of Monospecific Antibodies.

Every protein carries many antigenic determinants on its surface. As aresult, the immune response to such a molecule is always stimulation ofmany B-lymphocytes and production of just as many antibody species.Knowledge of the amino acid sequence of such epitopes is of greatimportance for immunological research.

It would be advantageous for many applications if one could obtain largeamounts of monospecific antibodies from mapped serum immediately aftermapping of epitopes. Monospecific antibodies are equally monoclonal intheir properties, and are also sold commercially. To isolate suchantibodies, the peptides, which comprise the epitope, must be coupled toa matrix. Then the serum is passed over that matrix, and the antibodies,which bind specifically to the desired antigen, are eluted. In thisexample of the application of the Intimin-based bacterial surfaceexposure, epitope-presenting E. coli cells were used as such a matrix.

FIG. 9 shows a survey of the procedure in epitope mapping usingIntimin-mediated cell surface exposure. Example 2 presents the use ofthis process.

Isolation of Peptides with Affinity to a Specific Target Protein ThroughIntimin-based Surface Exposure of Combinatory Peptide Libraries

To check whether Intimin-based bacterial surface exposure is suitablefor isolating from a molecular collection of surface-exposed peptidevariants those which have affinity to a specified target protein, alibrary of variants of the cystine node protein EETI-II, comprising 28amino acids, was generated. EETI-II is an inhibitor of trypsin proteaseswhich occurs in the vegetable marrow Ecballium elaterium. This peptideis stabilized by three intramolecular disulfide bridged which spread outa group of surface loops (described in Wentzel et al. (1999), J. Biol.Chem. 274: 21037–21043). A library of EETI-II variants was generated, inwhich the residues of two loop regions exposed to the solvent arerandomized. Example 3 presents the experimental procedure for thisexample embodiment.

The invention is explained in more detail in the following using someexperimental examples which will make it easier to understand theinvention, but which are not intended to limit the invention to theseexamples. Those skilled in the art will recognize which alternatives arepossible within the outlines of the invention on the basis of theseexamples.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures will serve for further explanation of theinvention. Individually, they show:

FIG. 1: Schematic representation of the structure of an Intimin. OM:external membrane; D0 to D3: extracellular domains 1 to 4. The Intiminfragment preferred here, which serves as the carrier for passengerdomains, lacks domains D2 and D3.

FIG. 2: Schematic representation of the expression vectorpASK-INT-EETI-CKSend. Intimin: coding sequence for the Intimin genefragment (SEQ ID NO: 17); etag: coding sequence for an epitope sequence(Etag); ead: coding sequence for the EETI-II microprotein; cat: gene forchloramphenicol acetyltransferase; tetR: gene for Tet repressor; bla:gene for β-lactamase. Corresponding translated amino acid sequence isSEQ ID NO:24.

FIG. 3: Flow-cytometric analysis of the bacterial surface exposure of anIntimin-EETI-II microprotein fusion protein.

FIG. 4: Survival proportions of E. coli cells which expose amicroprotein on the bacterial cell surface by means of the Intiminmembrane anchor.

FIG. 5: Schematic representation of control of gene expression throughsupE-mediated translation control.

FIG. 6 a: Nucleotide sequence and genetic organization of the sequencesegment of the E. coli genome fromBMH71-18 (SEQ ID NO:16) amplified bythe PCR primers SupE2-Eco-up (SEQ ID NO: 10) and SupE2-Mlu-lo (SEQ IDNO:11,complement). The tRNA coded genes are emphasized in bold type.

FIG. 6 b: Schematic representation of the vector pZA22-MCS1 (Lutz &Bujard (1997), Nucleic Acids Res. 25:1203) and of the vector pZA22-supEresulting from incorporation of the supE gene. PL-lac-O1: hybridoperator/promoter region from the PL operator and the lac promoter; RBS:ribosomal binding site; MCS-1: polylinker sequence; P15A: replicationorigin; KanR: Kanamycin resistance gene.

FIG. 7: Schematic representation of the vector pREP4-supE. Plac-supE:hybrid operator/promoter region of the PL operator and the lac promoter(Lutz & Bujard (1997) Nucleic Acids Res. 25:1203) followed by a sequencesegment containing the supE gene. P15A: replication origin; KanR:kanamycin resistance gene; lacI: gene for the lac repressor.

FIG. 8: Regulation of the synthesis and surface exposure ofIntimin-anchored passenger domains by controlled provision of supE-tRNA.

FIG. 9: Procedure for mapping of linear peptide epitopes.

FIG. 10: Representation of the two linker molecules resulting fromhybridization of 4 oligonucleotides, which generate the corresponding,vector cleavage sites (AvaI/BamHI). Coding strand of linker molecule onthe left, SEQ ID NO:18. Coding strand of linker molecule on the right,SEQ ID NO:19.

FIG. 11: Position of the epitope identified in the protein sequence ofPMS1. From left to right, epitope with sequence ECS . . . HYT, SEQ IDNO:20; epitope with sequence YFD . . . EKA, SEQ ID NO:21; epitope withsequence DSI . . . NSR, SEQ ID NO:22; epitope with sequence TRV . . .PWN, SEQ ID NO:23.

FIG. 12: A) PMS1_KL6 labeled with purified antibodies against clone 7(1) and with purified antibodies against its own epitope (2); B)PMS1_KL7 labeled with purified antibodies against clone 6 and withpurified antibodies against its own epitope (2).

FIG. 13: Amino acid sequence of the library of EETI-II variants (SEQ IDNO:7). Amino acids in the single letter code. The two loop regions inwhich amino acid positions (X) are randomized are indicated in italics

FIG. 14: Enrichment of the microproteins that bind the anti-β-lactamaseantibodies. All of the cells were labeled with anti-β-lactamase asdescribed in the text.

A) Unselected library labeled with anti-β-lactamase serum, anti-rabbitantibodies (biotin conjugate) and streptavidin-R phycoerythrin. 1.6% ofthe cells appear in the window used (fluorescence channels 300–1024). B)Second round of sorting (labeling as in A); C) Third round (labeling asin A).

FIG. 15: Amino acid sequence of the 6 EETI variants (1, 3, 5, 7, 8 and9), which interact with the anti-β-lactamase antibodies; randomizedamino acids are underlaid with gray. Underlines indicate a mutation fromthe wild type gene in the DNA sequence. A consensus sequence for bindingto the anti-underlaid with gray. Underlines indicate a mutation from thewild type gene in the DNA sequence. A consensus sequence for binding tothe anti-β-lactamase antibody is shown for the rear loop region. Variant1, SEQ ID NO: 1; Variant 3, SEQ ID NO: 5; Variant 5, SEQ ID NO: 2;Variant 7, SEQ ID NO: 16 Variant 8, SEQ ID NO: 3; Variant 9, SEQ ID NO:4.

FIG. 16A and B. A, sequence of amino acids 1 to 659 fromenterohemorrhagic Escherichia coli O157:H7 (SEQ ID NO: 25); B, sequenceof amino acids 1 to 753 from enterohemorrhagic Escherichia coli O157:H7(SEQ ID NO: 26).

EXAMPLES Example 1 Gene Fusion of a Nucleic Acid Sequence Segment Codingfor a Passenger Protein and One Coding for an Intimin Fragment VectorConstruction, and comparative Example

The cystine node polypeptide EETI-II, a trypsin protease inhibitorcomprising 28 amino acids, was selected as the passenger for the firstexample (as previously used by: Wentzel et al. (1999), J. Biol. Chem.274: 21037–21043; Christmann et al. (1999), Protein Eng. 12: 797–806).

For that purpose, the Intimin gene eae (Gene Bank Accession Z11541) fromEHEC O157:H7 Strain 933 bacteria was amplified with the polymerase chainreaction using the oligonucleotides Intiminup (SEQ ID NO:9) andIntiminlo (SEQ ID NO:8). EHEC O157:H7 bacteria inactivated by boilingwere used as the template. The gene was amplified with the followingamplification conditions: 30 sec 94° C., 30 sec 53° C. and 2 min 72° C.,30 cycles.

The oligonucleotide Intiminup hybridizes the eae gene from nucleotide 69to 88. This sequence segment contains the ATG start codon and the 17nucleotides downstream from it. This oligonucleotide contains 5′additional sequences which [represent/tr. note #6] cloning of the PCRproduct on the 5′ side into the Xbal cleavage site of the vectorpASK21TE-EETI-CKSend (Christmann et al. (1999), Protein Eng. 12:797–806). The oligonucleotide Intilo1 hybridizes the eae gene fromnucleotide 2028 to 2045 and contains at the 5′end the coding sequencefor a MluI cleavage site which allows cloning at the 3′ end in the EcoRIcleavage site of vector pAsk21-EETI. Thus the PCR product contains thecoding nucleic acid sequence for codons 1 to 659 of the Intimin. Then atripartite fusion gene in operative linkage with the tetApromoter/operator is localized in the resulting vector pASK-Inti-EETI.It consists of (a) the coding nucleic acid sequence for Intimin fromCodon 1 to 659; (b) the coding nucleic acid sequence for an epitope fromthe human Gla protein comprising 17 amino acids (Etag; Kiefer et al.(1990), Nucleic Acids res. 18: 1909) and (c) the coding nucleic acidsequence for the cystine node polypeptide EETI-CKSend (Christmann et al.(1999), Protein Eng. 12: 797–806; FIG. 2).

By cloning a PCR fragment, the CAG codon number 35 in Intimin wasreplaced by a TAG stop codon.

A second plasmid (pASK21TE-EETI-CKSend) in which the Intimin sequence isreplaced by a shortened OmpA fragment (Christmann et al. (1999), ProteinEng. 12:797–806) was used to be able to compare the expression levelwith previous processes for surface exposure.

The strain BMH71-18 (Christmann et al. (1999), Protein Eng. 12: 797–806)was used as the bacterial strain for the gene expression.

-   BMH71-18: [F¹laol^(q) lacZΔM15, proA+B+; Δ(lao-proAB), supE, thi]

The transformed cells were grown at 37° C. to an absorbance of 0.2 to0.4. Expression of the fusion gene was induced by addition of 0.2 ug/mlanhydrotetracycline. The surface exposure was demonstrated by binding ofa monoclonal anti-Etag antibody to the surface of the cells by means ofindirect fluorescence labeling. That was done by centrifuging the cellsoff after one hour of incubation and incubating them successively withmonoclonal anti-Etag antibody, biotinylated anti-mouse antibody andstreptavidin, phycoerythrin conjugate exactly as described by Christmannet al., (1999), Protein Eng. 12: 797–806. The labeled cells weremeasured in a flow cytometer (FIG. 3). The FACS diagram shows thefluorescence of cells which produce no fusion protein (negative control,A), those which produce the Intimin-Etag-EETI fusion protein (B) and,for comparison, (C) cells transformed with a plasmid in which theIntimin membrane anchor has been replaced by an Ipp-OmpA membrane anchorcorresponding to the previous Sder technique.

As can be seen from this figure, the cells in which Intimin-mediatedexposure of the fusion partner occurs exhibit about ten-fold higherfluorescence than the cells with the usual membrane anchor. This findingshows, surprisingly, that there are about ten times more molecules ofthe passenger protein available on the bacterial surface when theIntimin membrane anchor is used. Furthermore, the viability of thecells, which is strongly reduced in the previously described processesfor over-expression of membrane-anchored passenger proteins, is notnotably impaired by the overproduction of the fusion protein from theIntimin fragment and the passenger domain. The survival proportions ofthe cells before and 1, 2 and 4 hours after induction of the expressionof the fusion protein is shown below (FIG. 4).

Combined Use of supE tRNA in Host Organisms and the Amber Stop Codon inthe Intimin Fragment

The CAG codon number 35 in Intimin was replaced with a TAG stop codon bycloning a PCR fragment. When an amber suppressor strain is used, thisstop codon is over-read and a glutamine is incorporated at thatposition. As the efficiency of the amber suppression is low, fewermolecules are synthesized than in the absence of the stop codon. Thatmakes sure that the number of surface-exposed molecules remains withinan extent that is tolerable for the cell (in this respect, seeChristmann et al. (1999), Protein Eng. 12: 797–806). FIG. 2 shows thenucleotide sequence of the 5′-terminal and the ‘3’ terminal part of theIntimin gene in the expression vector pASK-INT-EETI-CKSend, with theamber stop codon #35 emphasized in bold face.

The amber suppressor strain BMH71-18 was used as the PCR template toobtain the supE.

-   BMH71-18: [F¹ laol^(q) lacZΔM15, proA+B+; Δ(lao-proAB), supE, thi]

The gene was amplified from the genome under standard conditions usingthe primers SupE2-Eco-up (SEQ ID NO:10) and SupE2-Mlu-lo (SEQ ID NO:11).FIG. 6 a shows the sequence of the genome segment and of the primer.

On page 15, please amend the fourth full paragraph as follows:

The two PCR primers SupE2-Eco-up (SEQ ID NO:10) and SupE2-Mlu-lo (SEQ IDNO:11) were used for amplification. The supE gene lies in a cluster oftRNA genes, which are transcribed polycistronically and then processedand liberated by specific RNAses. The supE gene and at least oneadditional tRNA gene upstream and downstream were amplified with theseoligonucleotides. The primers also introduce an EcoRI- and a MluIcleavage site. The resulting DNA segment was hydrolyzed with EcoRI andMluI and placed in the vector PZA22-MCS1 split with EcoRI and MluI (Lutz& Bujard (1997), Nucleic Acids Res. 25:1203), as shown in FIG. 6 b.

The sequence segment coding for the supE gene appears in this vector,again under the control of the P<lacO-1 promoter/operator MCS1 (Lutz &Bujard, Nucleic Acids Res. 1997, 25:1203) inducible with IPTG. Thepromoter/operator and the supE gene sequence segment downstream from itwere removed by splitting the resulting plasmid with XhoI and XabI. Theends were filled out with T4 polymerase in the presence of dATP, dGTP,dCTP and dTTP. The resulting fragment was inserted into the vectorpRep4, split with SmaI (Qiagen). FIG. 7 shows a schematic map of theresulting vector.

Transcription of the supE gene from pREP4-supE can be induced by IPTGinduction, thus inducing formation of supE-tRNA, which can be controlledby varying the quantity of the inducer. The plasmid pREP4-supE wasincorporated into the E. coli strain WK6 [(lac-proAB), thi, rpsL,nal^(r); F¹lacI⁴, lacZM15, proA+B+] to demonstrate ability to controlexpression of surface-exposed proteins. This strain has no chromosomallycoded supE gene. The strain was also transformed with the expressionplasmid pASK-INT-EETI-CKSend (see above). The E. coli strain, nowtransformed with two plasmids, was cultured in parallel trials in thepresence of increasing amounts of IPTG (0, 5, 10, 15, 20, 30, 40, 50,1000 μM). When an absorbance of 0.2 was reached, 0.2 μg/mlanhydracycline [tr. note #8] was added to induce transcription of theIntimin gene. The cells were harvested after one hour of induction. Thecells were labeled successively with anti-Etag antibody, biotinylatedgoat anti-mouse antibody and, finally, with phycoerythrin-coupledstreptavidin (see above). The fluorescence per cell which that causes isproportional to the number of Intimin-fusion proteins exposed on thesurface of the cell. The cellular fluorescence was measured by flowcytometry. As FIG. 8 shows, the intensity of the fluorescence per cell,and thus the number of exposed molecules, is directly correlated withthe amount of IPTG inducer. It can be modulated in other regions.

Example 2 Bacterial Surface Exposure of Protein Fragments, EpitopeMapping, and Isolation of Monospecific Antibodies

The gene for PMS1 from the yeast S. cerevisiae (Gene Bank AccessionNumber M29688) was used as the model antigen. The gene (2.7 kB) wasamplified using the PCR primers PMS1up (SEQ ID NO: 12) and PMS1lo (SEQID NO: 13) from S. cerevisiae. The DNA was purified with the NucleotrapPCR kit (Machery and Nagel). The DNAse I digestion was carried out infour different trial solutions using 1.8* 10⁻², 2,7^(?.), 10⁻², 3.6*10⁻² and 0.18 υ DNAse I [tr. note #9] in the presence of 2 mM MnCl2.5 μgDNA was used per solution. The restriction cutting was stopped byaddition of 500 mM EDTA after 10 minutes at room temperature. Theresulting fragments were separated on a 12.5% polyacrylamide gel afterstaining with ethidium bromide. Fragments in the size range of 40–100 bpwere cut out. Then the fragments were eluted from the gel by diffusionovernight in TBE buffer. Then any DNAse I that might still be presentwas removed by phenol/chloroform extraction and ethanol precipitation ofthe DNA. After that, overhanging ends were filled out with T4-DNApolymerase in the presence of dATP, dCTP, dGTP and dTTP. Thenoligonucleotide hybrid linkers (see FIG. 10) were ligated to them (2.5molar excess, 16 hours at 15° C.) and the ligation products were againpurified on a 12.5% [poly]acrylamide gel.

The solution conditions were the same as in the purification of the genefragments. Then the DNA was again extracted with phenol/chloroform andprecipitated. The cloning vector pASK-INT-EETI-CKSend was cut withAvaI/BamHI and purified by centrifugation in a sucrose gradient (Kolmar,H. & Fritz, H.-J. (1995). Oligonucleotide-directed mutagenesis withsingle-stranded cloning vectors. In: DNA Cloning 1: A PracticalApproach. D. Glover, B. D. Hames (Eds.), IRL Press, Oxford, pages193–224). A total of 230 ng split vector was ligated with the fragmentedDNA per gene bank, and electrocompetent BMH 71-18 cells were transformedwith it. A collection of 7,2* 10⁴ independent clones was obtained. Thepopulation of bacterial cells was incubated with polyclonal anti-PMS1rabbit serum after induction of the expression of the Intimin fusiongene by addition of anhydrotetracycline. The specific binding ofantibodies to epitope-carrying cells was determined by labeling withbiotinylated anti-rabbit antibodies and incubation withstreptavidin-phycoerythrin conjugate. Fluorescence-labeled cells weresorted by FACS and deposited individually on an agar plate with a MoFloCellsorter (Cytomation). DNA was prepared from a number of clones andthe nucleotide sequences of four cloned PMS1 gene fragments weredetermined. The DNA sequence of the clones involves the bases 259–330(PMS1_KL3), 1630–1672 (PMS1_KL6), 1786–1836 (PMS1_KL7) and 2590–2639(PMS1_KL10) of the pms1 gene (a total of 2715 bp). FIG. 11 shows thepositions of these epitopes in the peptide sequence of the PMS1 protein,as well as the translated amino acid sequence of the cloned PMS1sequence segment.

Cultures of the clones PMS_KL6 and PMS_KL7 were grown on the 50 mlscale. After one hour of induction by addition of anhydrotetracycline(02 μg/ml) at an absorbance of 0.2, the cells were pelleted, washed withPBS buffer, and resuspended in 500 μl PBS. Then 200 μl MS1 serum wasadded to these bacteria and the suspension was incubated on ice for 45minutes. Then the cells were pelleted and the supernatant was discarded.The cell pellet was resuspended in 200 μl glycine/NaCl (0.2 M glycine,0.145 M NaCl, pH 2.0), resuspended, and incubated for 45 minutes on ice.Following the incubation, the bacteria were centrifuged off. Thesupernatant was made alkaline by addition of 100 μl of 1 M Tris/HCl, pH9. The cells of clone KL6 and KL7 were incubated with the monospecificantibodies obtained in this manner (see FIG. 12).

This shows that monospecific antibodies can be isolated with thisprocedure.

Example 3 Isolation of Peptides with Affinity to a Specified TargetProtein by Intimin-based Surface Exposure of Combinatorial PeptideLibraries

A library of variants of the cystine node protein EETI-II, comprising 28amino acids, was generated. EETI-II is a trypsin protease inhibitor thatoccurs in the vegetable marrow Ecballium elaterium. This peptide isstabilized by three intramolecular disulfide bridges which produce aseries of surface loops (described in Wentzel et al. (1999), J. Biol.Chem. 274: 21037–21043). A library of EETI-II variants was generated, inwhich the groups of two loop regions exposed to the solvent arerandomized. The amino acid sequence of the EETI-II collection is:GCXXXXMRCKQDSDCLAGCVCQVLXPXXSXCG (SEQ ID NO:7). (Amino acids inone-letter code. The two loop regions in which amino acid position(x)sare randomized are indicated in italics.)

The randomized eeti genes were generated using PCR. An eeti-ckSend gene(see above) was used as the template. It was amplified using thedegenerate primer eti_(—)4+4up (SEQ ID NO:14) and eti_(—)4+4lo (SEQ IDNO:15). The randomization of the corresponding codons in the primersequence occurred according to the pattern NNS, in which N representsone of the four nucleotides and S represents G or C. This selectionexcluded the stop codons ochre (TAA) and opal (TGA) and reduced thenumber of possible codons from 64 to 32, but they still code for all theamino acids. The mixture of resulting PCR products was cut with AvaI andBamHI and ligated with the vector fragment pASK-INT-EETI-CKSend cut withthe AvaI/BamHI.

The pASK-INT-EETI-CKSend served as the cloning vector (see FIG. 8). Thisvector was cut with AvaI and BamHI. Then the vector fragment wasseparated from the vector DNA by means of sucrose gradientcentrifugation. The eeti4+4 genes generated by PCR with degeneratedprimers were ligated with the purified vector and the E. coli strainDH5((Hanahn, d. (1983), J. Mol. Biol. 166: 557–580) was transformed withthis solution. Sixteen transformations were done in parallel. All thetransformants were streaked on selective plates containingchloramphenicol (25 μg/ml). After incubation for about 20 hours at 37°C., the colonies were suspended and the cells were stored as aliquots at−80° C. after addition of DMSO (7%). A total of 2* 10⁷ independentclones was generated in this manner.

50 ml of liquid culture was inoculated with 10⁹ cells of the storedlibrary to isolate microproteins with affinity to anti-β-lactamaseantibodies. On reaching an absorbance of 0.4, anhydrotetracycline wasadded (0.2 μg/ml) to induce the gene expression. After induction, thecells were fluorescence-labeled. That was done by incubating the cellsfirst for 10 minutes anti-β-lactamase antibodies and then washing themwith PBS to remove unbound protein. Then the cells were incubated withbiotinylated anti-rabbit antibodies and then with streptavidin-coupledR-phycoerythrin. After labeling the cells were analyzed in theCellsorter, with fluorescent cells sorted out. Those bacteria which fellbetween 300 and 1024 in a fluorescence channel were defined asfluorescent (see FIG. 14).

Cells sorted out were plated on agar plates containing chloramphenicol(26 μg/ml) and incubated overnight at 37° C. The colonies were suspendedon the following day and stored as DMSO culture at −80° C. One aliquotwas used to inoculate a fresh 50 ml culture.

A total of 2* 10⁸ cells was analyzed in the first round. Those whichfell into the appropriate fluorescence channel (300–1024), were sortedout. In the second round, cells, which fell into this fluorescencechannel (4.2% in all), were sorted out and then immediately resortedtwice. These cells (5* 10⁵ events) were plated out and went to the thirdround on the next day. Here a significant increase of bacteria fallinginto the selected range was recorded (30%). The cells were again sortedand plated so that individual clones could be analyzed on the followingday. Fifteen of twenty individual clones showed interaction withanti-β-lactamase. Plasmid DNA was isolated from 6 of these clones, andthe nucleotide sequence of the gene for the EETI-CK variant wasdetermined (FIG. 15).

Protein sequences from the Intimin family usable within the limits ofthis invention are described at the following locations in theliterature:

-   1) Gamma Intimin (Escherichia coli), NCBI GI 3941710, NCBI GI    3941712, NCBI GI 3941714, McGraw, E. A., in “Molecular evolution and    mosaic structure of alpha, beta Intimins of pathogenic Escherichia    coli”, Mol. Biol. Evol. 16 (1) 12–22 (1999).-   2) Intimin (attaching and effacing protein, eae protein) NCBI GI    1169452, Yu, Ji and Kaper, J. B., in “Cloning and characterization    of the eae gene of enterohae. Escherichia coli O157:H7”, Mol.    Microbiol. 6(3), 411–417 (1992).-   3) eae gene, NCBI GI 384173, Beebakhee, G., et al. In “Cloning and    nucleotide sequence of the eae gene homologue enterohemorrhagic    Escherichia coli Serotype O157/H7”, FEMS Microbiol. Lett. 91(1),    63–68 (1992).-   4) Intimin (Escherichia coli), NCBI GI 2565325, Voss, E. et al. in    “Translocated intimin receptors (Tir) of shiga-toxigenic E. coli    isolates belonging to serogroups O26, O111 and O157 with sera from    patients with hemolytic-uremic syndrome and marked sequence    heterogeneity”, Infect. Immun. 66(11), 5580–5586 (1998).-   5) Intimin (Escherichia coli) NCBI GI 2865299, Elliott, S. J., et    al. In “The complete sequence of the locus of enterocyte effacement    from enteropathogenic Escherichia coli E2348/69”, Mol. Microbiol.    28(1), 1–4 (1998).-   6) Beta Intimin (Escherichia coli), NCBI GI 3941718, McGraw et al.,    loc. cit.-   7) Intimin (Escherichia coli), NCBI GI 2739264, Deibel, D. et al. In    “EspE, a novel secreted protein of attaching and effacing is    directly translocated into infected host cells, whereas a    trypsin-phosphorylated 90 kDa protein” [sic] Mol. Microbiol. 28(3),    463–474 (1998).-   8) Intimin (Escherichia coli) NCBI GI 4388530-   9) Intimin (Escherichia coli) NCBI GI 4388530-   10) Intimin type epsilon (Escherichia coli) NCBI GI 6683770-   11) Intimin NCBI GI 1947048-   12) Intimin NCBI GI 4106360-   13) Intimin (Escherichia coli) NCBI GI 6649538-   14) Intimin (Escherichia coli) NCBI GI 2809548-   15) Intimin (Escherichia coli) NCBI GI 7384863-   16) Shares homology with the enteropathogenic E. coli (EPEC)    attaching and effacing) gene, putative NCBI GI 304362-   17) Intimin (Escherichia coli) NCBI GI 7384863.    Intiminup:-   5′- GCGCTCTAGATAACGAGGGAAAAAATGATTACTCATGGTTGTTATAC -3′ (SEQ ID    NO:9)    Intilo 1:-   5′- GCGCCAATTGCGCTGGCCTTGGTTTGATC -3′ (SEQ ID NO: 8)    SupE2_Ecoup-   5′- GCGCGAATTCACCAGAAAGCGTTGTACGG -3′ (SEQ ID NO:10)    SupE2-Mlu-lo-   5′-GCGCACGCGTAAGACGCGGCAGCGTCGC -3′ (SEQ ID NO:11)    PMS1up-   5′-GCGATGTTTCACCACATCG -3′ (SEQ ID NO: 12)    PMS1lo:-   5′-TCATATTTCGTAATCCTTC- 3′ (SEQ ID NO:13)    eti-4+4up:-   5′-GACGCCCGGGTGCNNSNNSNNSNNSATCCGTTGCAAACAGGACTCCG -3′ (SEQ ID    NO:14)    eti-4+4lo:-   5′GCGCGCGGATCCGCASNNAGASNNSNNAGGSNNGAGAACCTGGCAAACGCAGCCA GCCAG -3′    (SEO ID NO:15)

1. A process for exposing peptides and proteins on the surface of hostbacteria, comprising the steps of (a) preparing a Gram-negative hostbacterium that is transformed with a vector on which a fused nucleicacid sequence is localized which is in operative linkage with anexogenously inducible promoter, wherein said fused nucleic acid sequencecodes for (i) a membrane anchoring domain which is an Intimin shortenedby at least the D3 domain in the carboxyterminal region, and (ii) aheterologous passenger peptide or passenger polypeptide to be exposed onthe surface of the host bacterium, and (b) cultivating the hostbacterium under conditions in which the heterologous passenger peptideor passenger polypeptide is expressed and exposed on the surface of thehost bacterium.
 2. A process according to claim 1, wherein the shortenedIntimin membrane anchoring domain is shortened by at least one otherIntimin domain selected from the group consisting of D0, D1, and D2. 3.A process according to claim 1, wherein the shortened Intimin membraneanchoring domain is from Enterobacteriaceae and the host bacterium is anEnterobacteriaceae.
 4. A process according to claim 3, wherein theshortened Intimin membrane anchoring domain is from enterohemorrhagicEscherichia coli.
 5. A process according to claim 4, wherein saidenterohemorrhagic Escherichia coli is Escherichia coli O157:H7.
 6. Aprocess according to claim 5, wherein the shortened Intimin membraneanchoring domain contains amino acids 1 to 659 (SEQ ID NO: 25) ofEscherichia coli O157:H7 Intimin.
 7. A process according to claim 5,wherein the shortened Intimin membrane anchoring domain contains aminoacids 1 to 753 (SEQ ID NO: 26) of Escherichia coli O157:H7 Intimin.
 8. Aprocess according to claim 1, in which expression of the fused nucleicacid sequence can be controlled, whereby (i) a codon of a nucleic acidsequence coding for the shortened Intimin membrane anchoring domainwhich codes for glutamine is replaced by an amber stop codon (TAG), and(ii) an Escherichia coli host strain is used in which translation of themRNA of the fused nucleic acid sequence is accomplished by providing acontrollable amount of suppressor tRNA which allows over-reading of thestop codon in the translation.
 9. A process according to claim 8, inwhich regulation of the expression of a gene for the amber suppressortRNA takes place due to the fact that it is placed under control of apromoter which is controllable in its transcription rate.
 10. A processaccording to claim 9, in which the promoter, which is controllable inits transcription rate, is the PLlac promoter.
 11. A process forproducing a variant population of surface-exposedpeptides/polypeptides/proteins with a desired property in host bacteria,and identifying host bacteria which expose the passengerpeptide/polypeptide/protein stably on their surfaces, comprising thesteps of: (i) producing one or more fusion genes or fusion genefragments by cloning a coding sequence of a passengerpeptide/polypeptide/protein in a continuous reading frame with a codingsequence of a shortened Intimin gene in at least one expression vector,wherein said shortened Intimin is shortened by at least the D3carboxybterminal domain; (ii) varying the passengerpeptide/polypeptide/protein by one of the methods selected from:intentional site-directed mutagenesis through the polymerase chainreaction (PCR) using oligonucleotides with intentionally exchangedbases, by random mutagenesis using oligonucleotides with randomlygenerated base sequences in selected sequence segments in the PCR,through error-prone PCR, randomly controlled chemical andradiation-generated mutagenesis, (iii) introducing the at least oneexpression vector into the host bacteria which expose the passengerpeptide/polypeptide/protein stably on their surfaces, (iv) expressingthe one or more fusion genes or fusion gene fragments in the hostbacteria, (v) cultivating the bacteria to produce a stablesurface-exposed passenger peptide/polypeptide/protein and, (vi)identifying host bacteria which carry a passengerpeptide/polypeptide/protein with a desired property on their surface.12. A process according to claim 11, which further comprises: (vii)selective enrichment of the host bacteria which carry the passengerpeptide/polypeptide/protein with the desired property on their surface.13. A process according to claim 11 in which the identification ofbacteria which carry a passenger peptide/polypeptide/protein with adesired binding affinity exposed on the surface is accomplished bybinding to a binding partner that is immobilized or labeled, or both.14. A process according to claim 11, in which a population of bacteriawhich carry a surface-exposed passenger peptide/polypeptide/protein witha binding affinity to a binding partner is used as a matrix for affinitychromatography purification of binding partners from a mixture ofsubstances.
 15. An expression vector for the expression of a fusion geneunder the control of an exogeneously inducible promoter in which thereis, in operative linkage with the promoter, a coding sequence for adesired passenger peptide in a continuous reading frame downstream to acoding sequence for an Intimin membrane anchoring domain, wherein saidIntimin membrane anchoring domain is shortened by at least the D3 domainin the carboxy-terminal region of 280 amino acids.
 16. An expressionvector according to claim 15, in which the promoter is selected from thegroup consisting of lac promoter, ara promoter, and tetA promoter.
 17. Agram-negative host bacterium of the family Enterobactericeae,transformed with at least one expression vector according to claim 15.18. A process according to claim 12, in which the identification ofbacteria which carry a passenger peptide/polypeptide/protein with adesired binding affinity exposed on the surface is accomplished bybinding to a binding partner that is immobilized or labeled, or both.19. A process according to claim 12, in which a population of bacteriawhich carry a surface-exposed passenger peptide/polypeptide/protein witha binding affinity to a binding partner is used as a matrix for affinitychromatography purification of binding partners from a mixture ofsubstances.